Plasma and tissue levels of High Mobility Group Box Protein 1 (HMGB1), a nuclear non-histone DNA-binding protein, are elevated for prolonged periods in chronic and acute inflammatory conditions, such as those associated with hemorrhage, trauma, or sepsis. Blockade of HMGB1 reduces organ dysfunction and improves survival in these pathophysiologic conditions. While HMGB1 was initially postulated to have potent proinflammatory properties and to contribute to acute lung injury, multiple organ dysfunction, and mortality by directly activating macrophages and other cellular populations, results from our laboratory and others have demonstrated that HMGB1 itself does not induce macrophages to produce cytokines or other proinflammatory mediators. In recent studies, we found that HMGB1 inhibits phagocytosis of apoptotic neutrophils by macrophages in vivo and in vitro. Phagocytosis of apoptotic cells, also called efferocytosis, is an essential feature of immune responses and is critical to resolution of inflammation. Our findings therefore describe a novel mechanism through which HMGB1 may be involved in enhancing inflammatory responses and contributing to the development, perpetuation, and severity of organ dysfunction associated with severe infection, blood loss, or trauma. Our hypothesis is that: HMGB1, by acting as a don't eat me signal that directly affects the accumulation and clearance of neutrophils in the lungs and other organs, is centrally involved in determining the development, perpetuation, and severity of hemorrhage and sepsis induced organ dysfunction. Our specific aims are: 1) To determine the mechanisms through which HMGB1 modulates phagocytosis and clearance of neutrophils by identifying the receptors on macrophages that are engaged by HMGB1 and that participate in modulating phagocytosis of apoptotic neutrophils, delineating the effects of HMGB1 in modifying the expression of receptors and ligands involved in phagocytosis of neutrophils, examining alterations in intracellular signaling pathways that occur in macrophages exposed to apoptotic neutrophils associated with HMGB1, and measuring the expression of pro- and anti-inflammatory cytokines by macrophages after ingestion of apoptotic neutrophils either associated with or not associated with HMGB1; 2) To examine the role of HMGB1 binding co-factors, including IL-12, LPS, TNF-1, and bacterial DNA, in modulating the ability of HMGB1 to affect phagocytosis of apoptotic neutrophils and activation of macrophages during efferocytosis; and 3) To determine the in vivo mechanisms through which HMGB1 contributes to the development and severity of acute lung injury, by identifying the importance of HMGB1 in modulating phagocytosis of apoptotic neutrophils in the lungs, delineating how interactions between HMGB1 and proinflammatory mediators affect phagocytosis of apoptotic neutrophils in the lungs, and examining the importance of HMGB1 in modulating phagocytosis of apoptotic neutrophils in the lungs during hemorrhage, TLR2, or TLR4 induced acute lung injury. The proposed studies should not only improve understanding of cellular mechanisms leading to ALI and organ system failure after hemorrhage, severe trauma, and sepsis, but also are likely to suggest novel therapeutic interventions aimed at decreasing the incidence and/or severity of organ system dysfunction and improving survival in patients with these life-threatening conditions. PUBLIC HEALTH RELEVANCE: Description Plasma and tissue levels of High Mobility Group Box Protein 1 (HMGB1) are elevated for prolonged periods in chronic and acute inflammatory conditions. Our recent findings, showing that HMGB1 can diminish the uptake and clearance of neutrophils by macrophages, suggest a novel mechanism through which HMGB1 participates in the development of organ failure after hemorrhage, trauma, and life-threatening infection. The studies proposed in this application should not only improve understanding of cellular mechanisms leading to organ dysfunction and death after trauma, hemorrhage, or sepsis, but also are likely to suggest novel therapeutic interventions aimed at improving outcome for patients suffering from these clinical problems.